Activation of AMP-protein activated kinase by oxaloacetate compounds

ABSTRACT

The present invention relates to oxaloacetate compounds that activate AMP-activated protein kinase (AMPK), including the preparation of the compounds, compositions containing the compounds, preserving said compounds and the use of the compounds in the prevention or treatment of disorders such as diabetes, metabolic syndrome, obesity, cardiovascular disease, Alzheimer&#39;s disease, and cancer.

This is a divisional of Application Ser. No. 13/806,465 filed Feb. 19,2013, which is a 35 U.S.C. §371 National Stage application ofInternational Application No. PCT/US2011/041377 filed Jun. 22, 2011,claiming priority based on U.S. Provisional Application No. 61/357,263filed Jun. 22, 2010, the contents of all of which are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

AMP-protein activated Kinase (AMPK) is a sensor and regulator ofcellular energy homeostasis, is a master switch regulating glucose andlipid metabolism and activation of AMPK results in many beneficialeffects. (Misra, et al, The role of AMP kinase in diabetes, Indian J MedRes 125:389-398 (2007); Kola, et al, The Role of AMP-Activated ProteinKinase in Obesity, Obesity and Metabolism, Vol 36 (2008); Kahn, et al,AMP-activated protein kinase: ancient energy gauge provides clues tomodern understanding of metabolism. Cell Metab. 1(10:15-25 (2005);Hardie, D. G. and Hawley, S. A. AMP-activated protein kinase: the energycharge hypothesis revisited. Bioessays 23: 1112 (2001), Kemp, B. E.et.al. AMP-activated protein kinase, super metabolic regulator. Biochem.Soc. Transactions 31:162 (2003)). AMPK can be activated by threedistinct mechanisms; 1) allosteric activation, 2) stimulation ofphosphorylation of the alpha-subunit on Thr172 by upstream kinase(s),and 3) inhibition of dephosphorylation by protein phosphatases (Kola, etal, The Role of AMP-Activated Protein Kinase in Obesity. Obesity andMetabolism 36:198-211 (2008)). The resulting activation leads to adecrease in fatty acid synthesis and oxidation, and a decrease incholesterol synthesis (Carling, D. et.al. A common bicyclic proteinkinase cascade inactivates the regulatory enzymes of fatty acid andcholesterol biosynthesis. FEBS Letters 223:217 (1987)). Other effects ofAMPK activation include positive changes in the levels of potential drugtargets for components of the metabolic syndrome including hormonesensitive lipase, glycerol-3-phosphate acyltransferase, malonyl-CoAdecarboxylase, and hepatocyte nuclear factor-4.alpha. AMPK activationstimulates glucose transport in skeletal muscle and controlsexpressional regulation of key genes in fatty acid and glucosemetabolism in liver (summarized in U.S. patent application Ser. No.7,119,205).

Genomic pathways that AMPK activation affects include decreasedexpression of glucose-6-phosphatase (a key enzyme in hepatic glucoseproduction) (Lochhead, P. A. et.al. 5-aminoimidazole-4-carboxamideriboside mimics the effects of insulin on the expression of the 2 keygluconeogenic genes PEPCK and glucose-6-phosphatase. Diabetes 49:896(2000)), and SREBP-1c (Zhou, G. et.al. Role of AMP-activated proteinkinase in mechanism of metformin action. The J. of Clin. Invest. 108:1167 (2001)), a key lipogenic transcription factor. Note that themetabolic changes induced by AMPK activation are both acute changes dueto phosphorylation of key enzymes, and longer-term effects on theexpression of genes involved in metabolic regulation.

There have been several studies that indicate that activation of AMPKwill result in many benefits. In the liver, there is decreased glucoseoutput and improvement in glucose homeostasis, decreased fatty acid andcholesterol synthesis and increased fatty acid oxidation. In skeletalmuscle tissue there is increased glucose uptake and fatty acidoxidation. There is a reduction in intra-myocyte triglycerideaccumulation and improved insulin action. A reduction in the ability tostore fat, due to the down-regulation of fatty acid synthesis, resultsin long-term weight reductions. The combinations of all these effectsare an excellent treatment for metabolic syndrome, diabetes and obesity.

Several studies in rodents and humans support that AMPK activation leadsto substantial benefits (Bergeron, R. et.al. Effect of5-aminoimidazole-4-carboxamide-1(beta)-D-ribofuranoside infusion on invivo glucose metabolism in lean and obese Zucker rats. Diabetes 50:1076(2001), Song, S. M. et.al. 5-Aminoimidazole-4-darboxamide ribonucleosidetreatment improves glucose homeostasis in insulin-resistant diabeted(ob/ob) mice. Diabetologia 45:56 (2002), Halseth, A. E. et.al. Acute andchronic treatment of ob/ob and db/db mice with AICAR decreases bloodglucose concentrations. Biochem. and Biophys. Res. Comm. 294:798 (2002),Buhl, E. S. et.al. Long-term AICAR administration reduces metabolicdisturbances and lowers blood pressure in rats displaying feature of theinsulin resistance syndrome. Diabetes 51: 2199 (2002)). Activation ofAMPK increases mitochondrial biogenesis (Reznick, et al, The role ofAMP-activated protein kinase in mitochondrial biogeneses. J Physiol574.1 (2006)). Reduced mitochondria content is important in thepathogenesis of insulin resistance and type 2 diabetes.

Many in vivo studies have relied on the AMPK activator5-Aminoimidazole-4-darboxamide ribonucleoside (AICAR), a cell permeableprecursor of ZMP. ZMP acts as an intracellular AMP mimic, and, whenaccumulated to high enough levels, is able to stimulate AMPK activity(Corton, J. M. et.al. 5-Aminoimidazole-4-carboxamide ribonucleoside, aspecific method for activating AMP-activated protein kinase in intactcells? Eur. J. Biochem. 229: 558 (1995)). Several in vivo studies havedemonstrated beneficial effects of both acute and chronic AICARadministration in rodent models of obesity and type 2 diabetes(Bergeron, R. et.al. Effect of5-aminoimidazole-4-carboxamide-1(beta)-D-ribofuranoside infusion on invivo glucose metabolism in lean and obese Zucker rats. Diabetes 50:1076(2001), Song, S. M. et.al. 5-Aminoimidazole-4-darboxamide ribonucleosidetreatment improves glucose homeostasis in insulin-resistant diabeted(ob/ob) mice. Diabetologia 45:56 (2002), Halseth, A. E. et.al. Acute andchronic treatment of ob/ob and db/db mice with AICAR decreases bloodglucose concentrations. Biochem. and Biophys. Res. Comm. 294:798 (2002),Buhl, E. S. et.al. Long-term AICAR administration reduces metabolicdisturbances and lowers blood pressure in rats displaying feature of theinsulin resistance syndrome. Diabetes 51: 2199 (2002)). For example, 7week AICAR administration in the obese Zucker (fa/fa) rat leads to areduction in plasma triglycerides and free fatty acids, an increase inHDL cholesterol, and a normalization of glucose metabolism as assessedby an oral glucose tolerance test (Minokoshi, Y. et.al. Leptinstimulates fatty-acid oxidation by activating AMP-activated proteinkinase. Nature 415: 339 (2002)). In both ob/ob and db/db mice, 8 dayAICAR administration reduces blood glucose by 35% (Halseth, A. E. et.al.Acute and chronic treatment of ob/ob and db/db mice with AICAR decreasesblood glucose concentrations. Biochem. and Biophys. Res. Comm. 294:798(2002)). In addition to AICAR, more recently it was found that thediabetes drug metformin can activate AMPK in vivo at high concentrations(Zhou, G. et.al. Role of AMP-activated protein kinase in mechanism ofmetformin action. The J. of Clin. Invest. 108: 1167 (2001), Musi, N.et.al. Metformin increases AMP-activated protein kinase activity inskeletal muscle of subjects with type 2 diabetes. Diabetes 51: 2074(2002)).

In addition to pharmacologic intervention, several transgenic mousemodels have been developed in the last years, and initial results arebecoming available. Expression of dominant negative AMPK in skeletalmuscle of transgenic mice has demonstrated that the AICAR effect onstimulation of glucose transport is dependent on AMPK activation (Mu, J.et.al. A role for AMP-activated protein kinase in contraction andhypoxia-regulated glucose transport in skeletal muscle. Molecular Cell7: 1085 (2001)).

Lowering of blood pressure has been reported to be a consequence of AMPKactivation (Buhl, E. S. et.al. Long-term AICAR administration reducesmetabolic disturbances and lowers blood pressure in rats displayingfeature of the insulin resistance syndrome. Diabetes 51: 2199 (2002)),therefore activation of AMPK has beneficial effects in hypertension.Through combination of some or all of the above-mentioned effectsstimulation of AMPK is expected to reduce the incidence ofcardiovascular diseases (e.g. MI, stroke). Endothelial NO synthase(eNOS) has been shown to be activated through AMPK mediatedphosphorylation (Chen, Z.-P., et.al. AMP-activated protein kinasephosphorylation of endothelial NO synthase. FEBS Letters 443: 285(1999)), therefore AMPK activation by any means can be used to improvelocal circulatory systems.

Increased fatty acid synthesis is a characteristic of many tumor cells,therefore decreased synthesis of fatty acids through activation of AMPKcan be useful as a cancer therapy. Cell cultures exposed to AICAR toactivate AMPK attenuated the growth of MDA-MB-231 tumors in nude mice(Swinnen, JV, et al. Mimicry of a Cellular Low Energy Status BlocksTumor Cell Anabolism and Suppresses the Malignant Phenotype. Cancer Res2005; 65:6 (2005)). The AMPK activator metformin inhibits breast cancercells, pancreatic cancer (Zakikhani, et. at, Metformin is anAMP-Kinase-Dependent Growth Inhibitor for Breast Cancer Cells. CancerResearch 66, 10269-10273 (2006), (Schhneider, et. al, Metformin clearlyinhibits pancreatic cancer. Cancer Detection and Prevention Online,Abstract 260 (2002)) and reduces overall cancer risk (Evans, et al,Metformin and reduced risk of cancer in diabetic patients. BMJ330:1304-1305 (2005)). Resveratrol also activates AMPK to kill cancercells (Hwang, et. al. Resveratrol Induces Apoptosis in ChemoresistantCancer Cells via Modulation of AMPK Signaling Pathway. Annals of the NewYork Academy of Sciences, V 1095 Signal Transduction Pathways, Part C:Cell Signaling in Health and Disease, 441-448 (2007)).

There are several current methods to activate AMPK, but these methodshave some problems associated with them. As stated above, AMPK can beactivated with metformin, resveratrol and AICAR. But metformin may alsoproduce lactic acidosis, which can become a life-threatening condition,especially where a patient has renal insufficiency. Still further,metformin therapy is often counter-indicated where a patient takes otherdrugs that interfere with renal function. Resveratrol has a very lowbioavailability, and large amounts may be necessary in order to achieveefficacy. AICAR is banned by the World Anti-Doping Code for athleticevents (The Prohibited List 2009, World Anti-Doping Agency,http://www.wada-ama.org/rtecontent/document/2009_Prohibited_List_ENG_Final_20_Sept_08.pdf),and is currently not available for human use.

Other methods to activate AMPK include diabetic drug classthiazolidinediones (LeBrasseur, et al, Thiazolidinediones can rapidlyactivate AMP-activated protein kinase (AMPK) in mammalian tissues. Am JPhysiol Endocrinol Metab (2006) and (Matejkova, et al, Possibleinvolvement of AMP-activated protein kinase in obesity resistanceinduced by respiratory uncoupling in white fat. FEBS Letters,539(1-3):245-248 (2003)). However, various thiazolidinediones have beenwithdrawn from the market or development has discontinued due torelatively high hepatotoxicity. (Isley, Hepatotoxicity ofthiazolidinediones. Expert Opinion on Drug Safety, 2(6):581-586 (2003)).Recently, overstimulation of PPAR gamma has also been implicated inincreased chances of developing colorectal cancer. The withdrawal oftroglitazone has led to concerns of the other thiazolidinediones alsoincreasing the incidence of hepatitis and potential liver failure, anapproximately 1 in 20,000 individual occurrence with troglitazone.Because of this, the FDA recommends two to three month checks of liverenzymes for the first year of thiazolidinedione therapy to check forthis rare but potentially catastrophic complication. In addition,thiazolidinediones have a side effect of water retention, leading toedema. Recent studies have shown there may be an increased risk ofcoronary heart disease and heart attacks with the thiazolidinedionerosiglitazone (Clinical Trials for Rosiglitazone,www.ClinicalTrials.gov).

The reduction of calories below baseline ad. Librium feeding levels(Calorie Restriction) is one method to induce AMPK activation. Theextension of lifespan in the nematode worm Caenorhabditis elegans areshown to be completely dependent on the activation (phosphorylation) ofAMP-protein activated kinase (AMPK) and the FOXO transcription factorDAF-16 (Greer, et al, An AMPK-FOXO pathway mediates longevity induced bya novel method of dietary restriction in C. elegans. Current Biology9:17(19):1646-56 (2007)). Under Calorie Restriction, cellular energydepletion causes rising AMP levels, and an increase in the NicotinamideAdenine Dinucleotide (NAD+) level as compared to the reduced level(NADH), results in activation of AMPK (Raphaloff-Phail, et alBiochemical regulation of mammalian AMP-activated protein kinase (AMPK)activity by NAD and NADH. Journal of Biological Chemistry, ManuscriptM409574200 (2004)). While Calorie Restriction is a low-risk method toactivate AMPK, it requires the reduction of baseline food consumption tolevels that are not desirable to the majority of the population due toextreme hunger. The diet is extremely difficult to follow over time.

Strenuous exercise can also activate AMPK (Lee-Young, et al “AMPKactivation is fiber type specific in human skeletal muscle: effects ofexercise and short-term exercise training, Journal of AppliedPhysiology, 2009 July; 107(1); 283-9) however, again, this may not bedesirable or achievable by the majority of the population.

In animal models, various stresses such as oxidative stress, hypoxia,ischemia and heat shock can activate AMPK (Towler, et. al, AMP-activatedprotein kinase in metabolic control and insulin signaling Circ Res. 100,328-341 (2007). These stresses are not recommended as an AMPK activationagent.

Undoubtedly, activation of AMPK can aid in the prevention or treatmentof disorders such as diabetes, metabolic syndrome, obesity,cardiovascular disease, dyslipidemia and cancer. Current activation ofAMPK which includes strenuous exercise, calorie restriction,pharmacological interventions mimetics metformin and resveratrol, andthiazolidinediones all have side effects which are detrimental ordifficult to implement over time. Therefore, there exists a need in themarket for an AMPK activator which has high bioavailability, lowtoxicity, and preferably is already a human metabolite which would loweroverall pharmacological risk. The currently proposed invention meetsthose needs.

SUMMARY OF THE INVENTION

In CR a low energy state produces signaling events to affect expressionlevels of genes involved with cellular proliferation, apoptosis,electron transport chain, immune response, protein turnover and proteinsynthesis (Lee C K, Pugh T D, Klopp R G, et al. The impact ofalpha-lipoic acid, coenzyme Q10 and caloric restriction on life span andgene expression patterns in mice. Free Radic Biol Med 2004;36:1043-57.).Identifying these signaling events that produce the CR metabolic statehas generated the search for CR mimetics, that is, compounds that invokethis state and produce the benefits of CR without actually requiring areduction in caloric intake (Lane M A, Ingram D K, Roth G S. The serioussearch for an anti-aging pill. Sci Am 2002;287:36-41.; also Ingram D K,Roth G S, Lane M A, et al. The potential for dietary restriction toincrease longevity in humans: extrapolation from monkey studies.Biogerontology 2006;7:143-8.). Interestingly, it appears there aremultiple longevity pathways that can be activated in CR, depending onhow CR is implemented (Greer, et al, An AMPK-FOXO pathway mediateslongevity induced by a novel method of dietary restriction in C.elegans. Current Biology 9:17(19):1646-56 (2007)). One such pathway,based on activating AMP-activated protein kinase (AMPK) and a functionaltranscription factor FOXO/DAF-16, has been directly tied to CR in C.elegans (Greer, et al, An AMPK-FOXO pathway mediates longevity inducedby a novel method of dietary restriction in C. elegans. Current Biology9:17(19):1646-56 (2007)). AMPK has also been shown to be activated underCR in animal models (Pallottini V, Montanari L, Cavallini G, BergaminiE, Gori Z, Trentalance A. Mechanisms underlying the impaired regulationof 3-hydroxy-3-methylglutaryl coenzyme A reductase in aged rat liver.Mech Ageing Dev 2004;125:633-9; also Greer, et al, An AMPK-FOXO pathwaymediates longevity induced by a novel method of dietary restriction inC. elegans. Current Biology 9:17(19):1646-56 (2007)). Both AMPK andFOXO/DAF-16 are conserved homologs throughout the animal kingdom, givingrise to the opportunity to use CR mimetics to activate AMPK as one ofthe CR pathways and receive the benefits of AMPK activation in mammals,including humans.

Previously, I discovered that oxaloacetic acid, its salts and precursorssuch as alpha-ketoglutarate and aspartate (“OAA” as a group) can act asCR mimentics (Cash, U.S. patent application Ser. No. 11/792,703 filedMay 13, 2008, which is a 371 of PCT/US05/46130 filed Dec. 15, 2005,claiming benefit of U.S. Provisional Application 60/637,287 filed Dec.17, 2004). I discovered that supplementation of the CR mimetic, OAA,activates AMPK and allows for the multiple benefits of AMPK activation,which is discussed herein.

AMPK is a heterotrimeric complex that consists of a catalytic alphasubunit and regulatory beta and gamma subunits that together make afunctional enzyme, conserved from yeast to humans. The alpha AMPKsubunit is activated in order to increase lifespan (Apfeld, et al, TheAMP-activated protein kinase AAK-2 links energy levels and insulin-likesignals to lifespan in C. elegans. Genes and Development 18:3004-3009(2004)). This same AMPK activation for lifespan extension has been shownto be the same mechanism as CR for lifespan extension (Greer, et al, AnAMPK-FOXO pathway mediates longevity induced by a novel method ofdietary restriction in C. elegans. Current Biology 9:17(19):1646-56(2007)).

I have assayed the CR mimetic OAA for AMPK activation by using the wormC. eleganscontaining a non-functional alpha AMPK subunit, and havecompared lifespan with and without the functional AMPK subunit. CRmimetics will increase the lifespan of C. elegans in worms withfunctional FOXO/DAF-16 and AMPK by 25%, p<0.001, but will not increasethe lifespan of the worms with a non-functional alpha AMPK subunit.Graph 1 is the Kaplan-Meyer survival curves for worms with functionaland non-functional AMPK showing the difference in lifespan. The graphsclearly show that OAA increases lifespan in the worms with functionalAMPK but do not increase lifespan in the worms with non-functional AMPK.This clearly indicates that the CR mimetic OAA increases AMPKactivation. Further, wild type worms with functional AMPK that aresubjected to calorie restriction do not have their lifespan increasedwith OAA supplementation, indicating that AMPK activation is a componentof the CR pathway.

With AMPK activation, there will be a reduction in the incidence, andbeneficial treatment of a variety of conditions including type 2diabetes, Alzheimer's disease, metabolic syndrome, obesity,cardiovascular disease, dyslipidemia, stroke and cancer.

I have investigated the toxicity of OAA in an acute study and twochronic studies. The acute study showed a LD50 of more than 5,000 mgOAA/Kg of mouse (when taken with food), generally considered to be theregulatory threshold for “non-toxic” compounds. The first chronic study,performed with 380 mg OAA/kg of mouse, performed over eighteen monthswith older mice, indicate a “negative” toxicity, as the mice on OAAlived 25% longer than the control group. In the second test, performedwith rats over 90 days, indicated no observed adverse effect level(NOAEL) of 500 mg OAA/kg of rat, the highest dose given in the study. Ashuman dosage for effective treatment is in the range of 50 mg to 2,000mg, more preferably from 100 mg to 1,000 mg, and most preferably from100 mg to 300 mg, per day, for a 70 kg person this is only a dosage of1.4 to 4.2 mg OAA /kg human. OAA shows a tolerance safety factor of 100×or more for treatment and prevention. This should not be surprising, asOAA is a key metabolite found in every human cell.

Thus, according to one embodiment of the present invention there isprovided a method of preventing and treating method of treating diabetesin a mammal, comprising administration of a therapeutically effectiveamount of OAA to activate AMPK.

In another embodiment of the present invention, there is provided apharmaceutical composition which includes a pharmaceutically acceptablecarrier in combination with OAA, effective to modulate glucosemetabolism in a mammal when the composition is administered to themammal at a concentration effective to modulate glucose metabolism.

Furthermore, it should be noted that contemplated pharmaceuticalcompositions may additionally include a second pharmaceutical agent, andmost preferably a pharmaceutical agent for treatment of type-2 diabetes,metabolic syndrome, pre-diabetes, and dyslipidemia. Thus, suitablesecond pharmaceutical agents include various biguanides, sulfonyl ureas,meglitinides, thiazolidinediones, and additional compounds used fortreatment.

In addition to contemplated pharmaceutical compositions for thetreatment of type-2 diatetes, metabolic syndrome, pre-diabetes anddyslipidemia, compositions with OAA may include nutritional supplementsthat are agents to treat these conditions, which include, but are notlimited to, caffeine, cinnamon, cinnamon extracts, oregano, Euonymusalatus, non-toxic chromium salts, Gymnema sylvestre, Alpha lipoic acid,Vanadyl sulfate, Biotin, B-complex vitamins, Vitamin C, Magnesium saltsand Magnesium stearate, resveratrol, CoQ10, Vitamin E, Banaba leaf,Thymus vulgaris extract, Pancreas extract, Adrenal extract, DHEA,Psyllium, Panax ginseng, Momordica charantia, Allium sativum, Vacciniummyrtillus, Trigonella foenum-gracecum, Gingko biloba, Oenothera biennis,peppermint, chamomile, passionflower, Corosolic acid, Pine Bark Extract(Proanthocyanidins).

In another aspect of the inventive subject matter, a method ofmodulating glucose metabolism in a mammal includes a step ofadministering OAA at a dosage effective to modulate glucose metabolismin the mammal, wherein the mammal is preferably diagnosed with at leastone of metabolic syndrome, pre-diabetes, type-2 diabetes anddyslipidemia. While not wishing to be bound by any specific theory orhypothesis, the inventors contemplate that the compounds according tothe inventive subject matter will modulate the glucose metabolism byincreasing AMPK activation through the use of OAA, and that suchactivation will modulate the glucose metabolism by increasing glucoseuptake in a muscle cell, and/or decreasing gluconeogenesis in ahepatocyte, along with stimulating the up-regulation of the FOXO3a geneassociated with glucose homeostasis.

In yet another aspect of the inventive subject matter, the inventorcontemplates a method of treating a condition in a mammal associatedwith dysregulation of AMPK, wherein the method comprises a step ofadministering one or more of contemplated compounds at a dosageeffective to activate AMPK, wherein the method comprises a step ofadministering OAA at a dosage effective to activate AMPK. Among otherdiseases, conditions associated with AMPK dysregulation includecardiovascular diseases, type 2 diabetes, and neoplastic diseases.

According to an additional embodiment of the present invention there isprovided a method of preventing and treating Alzheimer's disease in amammal, comprising administration of a therapeutically effective amountof OAA to activate AMPK.

According to an additional embodiment of the present invention there isprovided a method of preventing and treating metabolic syndrome in amammal, comprising administration of a therapeutically effective amountof OAA to activate AMPK.

According to an additional embodiment of the present invention there isprovided a method of preventing and treating obesity in a mammal,comprising administration of a therapeutically effective amount of OAAto activate AMPK.

According to an additional embodiment of the present invention, there isprovided a method of maintaining fat from re-accumulating after a diet,comprising administration of a therapeutically effective amount of OAAto activate AMPK.

According to an additional embodiment of the present invention, there isprovided a method of preventing and treating cardiovascular disease in amammal, comprising administration of a therapeutically effective amountof OAA to activate AMPK.

According to an additional embodiment of the present invention, there isprovided a method of preventing and treating cancer, comprisingadministration of a therapeutically effective amount of OAA to activateAMPK.

According to an additional embodiment of the present invention, there isprovided a method of increasing mitochondrial density, comprisingadministration of a therapeutically effective amount of OAA to activateAMPK.

According to an additional embodiment of the present invention there isprovided a pharmaceutical composition comprising a therapeuticallyeffective amount of OAA in combination with a pharmaceutically suitablecarrier to activate AMPK.

As set forth herein, the invention includes administering atherapeutically effective amount of OAA and prodrugs thereof to amammal. Preferably, the invention also includes administering atherapeutically effective amount of any OAA to a human to activate AMPK,and more preferably to a human in need of being treated for orprophylactically treated for any of the respective disorders set forthherein.

It is noted that the prevention and/or treatment of the disorders withOAA relates specifically to the activation of AMPK. The same disordersmay be treated with the calorie restriction mimetic OAA may be treatedthrough other pathways, which are covered in Cash, U.S. patentapplication Ser. No. 11/792,703 filed May 13, 2008, which is a 371 ofPCT/US05/46130 filed Dec. 15, 2005, claiming benefit of U.S. ProvisionalApplication 60/637,287 filed Dec. 17, 2004, incorporated herein byreference.

Stability of Oxaloacetic acid, oxaloacetate and oxaloacetate salts

Oxaloacetic acid (as opposed to OAA, previously defined as oxaloaceticacid, salts of oxaloacetic acid, metabolic precursors of oxaloaceticacid including alpha-ketoglutarate and aspartate and the oxaloacetateion) when dissolved in water ionizes to oxaloacetate. The oxaloacetatecan be in three forms depending on the pH of the solution. At low pH(<1.5) and low temperature (<4 degrees C.) oxaloacetate hydrates. Athigher pH, oxaloacetic acid in water occurs in three forms, 1) thehydrated form, 2) a keto form, and 3) an enol form. Outside of watersolutions, the solid form of oxaloacetic acid is primarily in the enolform. All forms of oxaloacetic acid and the ion oxaloacetate areabsorbed by the body. The hydrated form is mostly converted once itenters the higher pH of the body outside of the intestinal tract to theketo and enol form. As a specific example, at a pH of 6.9, oxaloaceticacid in water is composed of 5% in the hydrated form, 84% in the ketoform and 11% in the enol form. Enol-oxaloacetate is convert toketo-oxaloacetate with the enzyme enol-keto tautomerase, a ubiquitousenzyme throughout the human body.

While oxaloacetic acid can be given in any of the three forms and beeffective (because the forms change with different pH conditions andenzymatic activity), there is a significant problem with stability thathas prevented the commercialization of the compound as a therapeuticagent. Keto-oxaloacetic acid decarboxylates spontaneously into pyruvateand carbon dioxide, and neither byproduct of the decomposition iseffective in activating AMPK. The stability problems of oxaloacetic acidare well documented in the literature (Krebs, H A, “The effect ofinorganic salts on the ketone decomposition of oxaloacetic acid”,Biochem J. 1942, April; 36(3-4):303-5; also Ochoa, S. “Biosynthesis oftricarboxylic acids by carbon dioxide fixation; the preparation andproperties of oxalosuccinic acid”, J Biol Chem 1948 May; 174(1):115-22;also Lynen, F and Scherer, H. Ann. Chem. 560, 163(1948); also Kosicki,et al, “Lithium Chloride Catalyzed Decarboxylation of Oxalacetic Acid inEthanol”, Canadian Journal of Chemistry, Vol 42 (1964); also Kosicki andLipovac, “The pH and pD Dependence of the Spontaneous andMagnesium-ion-Catalyzed Decarboxylation of Oxalacetic Acid”, CanadianJournal of Chemistry, Vol 42 (1964); also Bontchev and Michaylova,“Cataytic Activity and Complexation I, Influence of Organic Solvents onthe Rate of Catalytic Decarboxylation of Oxalacetic Acid” J. Inorg.Nucl. Chem, 1967, Vol 29. Pp 2945 to 2953; also Kozlowske and Zuman,“Polarographic Reduction of Aldehyes and Ketones, Part XXX. Effects ofAcid-Base, Hydration-Dehyration and Keto-enol Equilibria on Reduction ofalpha-Ketoglutaric and Oxalacetic Acid and their Esters”, J.Electroanal. Chem, 226 (1987) 69-102) and Speck, John, “The effect ofCations on the Decarboxylation of Oxalacetic Acid” J. BiologicalChemistry, (1948)). The lack of stability of oxaloacetic acid has been asource of difficulty in the preparation of a commercial product.Yoshikawa (Yoshikawa, K, “Studies on Anti-diabetic Effect of SodiumOxaloacetate”, Tohoku J. Exp. Med, (1968) 96:127-141) teaches “Sinceoxaloacetic acid is unstable, its sodium salt was used in the animalexperiment as well as in the clinical investigations” (page 128).

The enol and keto form of oxaloacetic acid are tautomers, and in waterform a chemical equilibrium. At a pH of 6.9, oxaloacetic acid in wateris composed of 5% in the hydrated form, 84% in the keto form and 11% inthe enol form. The keto-oxaloacetate decarboxylates quickly to pyruvate.As the keto-oxaloacetate form disappears due to decarboxylation, theenol and hydrated form convert to keto-oxaloacetate, and then alsodecarboxylate into carbon dioxide and pyruvate, until all theoxaloacetate is consumed. Note that neither of the byproducts ofoxaloacetate decarboxylation, carbon dioxide and pyruvate, are effectivein activating AMPK. If there are divalent cations in the fluid, which isvery common, the decarboxylation of oxaloacetic acid can happen within aday.

Salts of oxaloacetic acid, have been tested and are also not stable,despite the teachings of Yoshikawa. As a specific example of this,Na-OAA when dissolved in water will form 5% hydrated form, 84% in theketo form and 11% in the enol form, similar to oxaloacetic acid in wateradjusted to a pH of 6.9. The keto-oxaloacetate decarboxylates quickly,followed by enol conversion to keto form and further decarboxylation.The sodium salt of oxaloacetic acid used in the Yoshikawa study musthave been made up daily, which is not practical for a commercialproduct. Na-OAA has no shelf life, and is not available commercially.

The hydrated form of oxaloacetic acid can be made stable by maintainingit at very low pH, but only for less than one-week's time, attemperatures not exceeding 8 degrees C. Again, this does not allowcommercial distribution of the product to persons needing prevention ortherapy gained from oxaloacetic acid supplementation. The hydrated formof oxaloacetic acid has no shelf life.

Methods to Stabilize Oxaloacetic Acid, Oxaloacetate and OxaloacetateSalts

In contrast with the multiple teachings of the literature, the currentinvention makes use of stable oxaloacetic acid in order to allow for areasonable shelf life of one year or more. The stable oxaloacetic acidactivates AMPK and achieves preventative and therapeutic effects. Theinvention uses anhydrous enol-oxaloacetic acid which is stable at roomtemperature for a period exceeding one year. The enol-oxaloacetic aciddoes not decarboxylate spontaneously and is thus stable if kept dry.Water catalyzes the equilibrium reaction between enol-and ketooxaloacetic acid. Note that only the keto form of oxaloacetic aciddecarboxylates into pyruvate and carbon dioxide spontaneously, not theenol-form. There is an energy gap between the enol and keto form whichis bridged when the compounds are exposed to water, however, this sameenergy gap prevents the conversion of the enol to keto form when theproduct is kept dry. Once there is a conversion to the keto form,decarboxylation can spontaneously occur at temperatures above thefreezing point of water. Thus, manufacturing oxaloacetate with a watercontent of less than 2% and keeping the oxaloacetic acid in a solidstate and dry through the use of moisture sealants and/or moistureabsorbents, creates the commercial shelf-stable enol-oxaloacetate form,even at room temperatures. Drying effectiveness can be increased byincreasing the drying time, drying under vacuum, by using anhydrouswashes of isopropyl alcohol or ethyl alcohol to absorb the remainingwater (and then evaporating the alcohol), or by performing multiplewashes with hexane or non-water soluble solvent to physically remove thewater, typically after an alcohol wash. The non-water soluble solventwould then be evaporated off. The small amount of non-water solublesolvent wash remaining in the oxaloacetic acid is non-toxic and servesto repel water moisture from entering into the powder to further extendshelf life. Hexane is a residual solvent in many commercial foodpreparations including decaffeinated coffee, and is not toxic in smallquantities. Alternatively, the final wash can be performed withliquefied propane, liquefied butane, ethyl acetate, ethane, carbondioxide, or nitrous oxide to reduce the water content.

The reduced water content oxaloacetate form can allow for the use of thecompound in commerce, as the shelf life of the product will exceed oneyear with less than 1% product decay at room temperatures. An example ofthe manufacture of oxaloacetic acid prior to the improved drying stepcan be seen in Bessman; “Preparation and Assay of Oxalacetic Acid”;Arch. Biochem., vol. 26, pp. 418-421, 1950, also in Heidelberger; “TheSynthesis of Oxalacetic Acid-1-C14 and Orotic Acid-6-C14”, Biochem.Prepn. 3, 59 (1953) pp 4704-4706.

In practice, the isolation of the oxaloacetate from water in theatmosphere can be easily achieved after encapsulation of the oxaloaceticacid by sealing the bottles or placing individual capsules in a plasticblister pack. Reducing the water content below 2%, and most preferablybelow 1% along with isolation from the atmosphere, will keep theoxaloacetate in the enol form, and will prevent decarboxylation.Additional measures to prevent decarboxylation include the use ofdesiccants in the container with the enol-oxaloacetate and the additionof 10% to 90% anhydrous ascorbic acid per weight of oxaloacetic acid, ormore preferably 50% anhydrous ascorbic acid per weight of oxaloaceticacid. Ascorbic acid acts as an electron acceptor and reduces the rate ofdecarboxylation. Most preferably, the combination of adding anhydrousascorbic acid, sealing the container, and using an enol-form oxaloaceticacid below a 1% moisture level combine to yield a shelf-life of theproduct at 30 degrees C. in excess of one year.

The present invention also describes the methods used to stabilizesodium oxaloactate (and other salts, solutions and buffered solutions ofoxaloacetic acid). Stabilization can be achieved by a biphasiccontainment system. Sodium oxaloacetate for commercial use can be madeby using the solid anhydrous enol-form and combining it with a solutionof water plus sodium hydroxide (NaOH) or other basic solution whenneeded. This can be in the form of a container with two separatecompartments, one that contains the basic solution, and one thatcontains the anhydrous enol-oxaloacetate separated by a breakablebarrier. When sodium oxaloacetate (or other salt) is needed, the barrierbetween the basic solution and the anhydrous oxaloacetate is broken, andoxaloacetate salt is quickly formed in solution. The solubility ofoxaloacetic acid in water is 100 mg/ml, allowing rapid digestion of theanhydrous enol-oxaloacetic acid. Specific applications of a biphasiccontainment system include a flexible capsule, such as a gel cap whichcan be compressed by hand or with teeth to break an inner seal betweenthe sodium hydroxide solution and the solid anhydrate enol-oxaloacetate.Another specific application of a biphasic containment system includesan intravenous (IV) bag with two compartments, one with an IV fluid andthe other with anhydrous enol-oxaloacetic acid separated by a breakablebarrier. When needed, the breakable barrier is ruptured, and the twocomponents are mixed. The IV fluid can be a buffered solution, anon-buffered solution, an acidic solution, a basic solution or a neutralsolution. Yet another example of a biphasic containment system is tohave two separate containers; one for the solid oxaloacetic acid, andone for the liquids. Two separate containers will allow the solidoxaloacetic acid to be placed in storage below 0 degrees C., while theliquid container is kept at a different temperature. Storing the dryoxaloacetic acid at −20 degrees C. will enable the use of commonlyavailable commercial oxaloacetic acid, without the additional dryingstep of the preparation. Again when needed, the two containers arejoined and mixed to yield the oxaloacetate salt solution. A list of thestorage temperature of commercially available oxaloacetic acid is shownbelow:

Storage Catalog Grade Assay Supplier temperature No. Oxaloacetic acid,puriss. 98.0-101.0% Reanal Private Ltd. below 10° C. 25380 Oxalaceticacid >=98.0% Research Organics Inc. −10-−25° C. 05130 Oxalaceticacid >=98.0% Chem-Impex below 0° C. 01460 International Oxalacetic Acid 98% MP Biomedicals 0° C. 100568 98-99% Oxalacetic Acid  98% MPBiomedicals 0° C. 194719 Cell Culture Reagent, 98-99% OxalaceticAcid >95% Wako Pure Chemical 2-10° C. 15-0041 Industries Oxalacetic acid≧98% Biosynth AG −15° C. O-5000 Oxaloacetic acid 97.5%-102.5% Sigma-Aldrich Corp. −20° C. Aldrich 171255 Oxaloacetic acid ^(~)98% ≧98%Sigma Aldrich Corp. −20° C. Sigma O4126 Oxaloacetic acid ≧98.0%   SigmaAldrich Corp. 2-8° C. Fluka 75660 BioChemika Oxaloacetic acid, ≧97.5%  Sigma Aldrich Corp. −20° C. Sigma O9504 Hybri-Max ™ powder, hybridomatested Oxaloacetic acid, ≧97% Sigma-Aldrich Corp. −20° C. Sigma O7753insect cell culture tested

Oxaloacetic Acid Effects in the Human Body

Humans that orally consume the solid oxaloacetic acid show reductions inblood glucose levels, due in part to activation of AMPK. The oxaloaceticacid is converted to the ionic form oxaloactate in the digestive tract,and is then absorbed into the body and is distributed throughout thebody via the bloodstream. Yoshikawa measured the rate of oxaloacetatedistribution into the bloodstream after 200 mg of sodium oxaloacetatewas given to normal and diabetic patients. At time 0, the amountdetected was 0 ug/100 ml blood, at time 30 minutes 0 ug/100 ml blood,and at time 60 minutes ranged from 1.5 to 3.1 mg/ 100 ml blood. Mystudies indicate that most of the oxaloacetic acid orally consumed ismoved from the blood stream and taken into individual cells within 120minutes. Within the cells the oxaloacetate reacts into L-malate as itencounters the enzyme malate dehydrogenase (MDH). MDH is ubiquitous inhuman cells, and the conversion of oxaloacetate to malate is veryenergetically favorable (the Gibbs Free Energy, delta G, is highlynegative (−29.7) indicating the reaction is very favorable to proceed).As oxaloacetate is converted to malate, NADH is converted to NAD+. Theresulting increase in the NAD+/NADH ratio leads to the phosphorylation(activation) of AMPK (Rafaeloff-Phail R, Ding L, Conner L, et al.“Biochemical regulation of mammalian AMP-activated protein Kinaseactivity by NAD and NADH”. J Biol Chem 2004; 279: 52934-9). AMPK servesto stimulate glucose uptake into the skeltal tissues and thereby reducesglucose levels. The immediate reduction is best seen with a glucosetolerance test in individuals that have defective leptin signaling(including overweight and obese persons) that are fasting. In theseindividuals, the defective leptin signaling which no longer activatesAMPK is corrected by the oxaloacetic acid supplementation and theresulting increase in the NAD+/NADH ratio and activation of AMPK.Additionally, older individuals have reduced uptake of glucose intotheir muscle tissues, even if they are of a lean body type. AMPKactivation can measureably improve glucose levels and glucose stabilitywith these people.

In addition to improvements in glucose levels and reduction in insulinresistance, continuing supplementation of oxaloacetic acidsupplementation and subsequent chronic NAD+/NADH ratio increase andfurther chronic activation of AMPK results in genomic changes. AMPK ispart of a signaling cascade that influences which genes expressproteins, and the amount of the expression. AMPK activation with thecalorie restriction mimetic OAA over a period of 2.5 weeks is sufficentto see genomic changes in mice liver tissues using gene chips, andsufficient to see glucose related effects in all body types in humans.Gene changes documented in mice in my previous patent applicationinclude an increase in FOXO3a by 100 to 200%. FOXO3a is a gene thathelps to regulate glucose levels. This increase in expression of FOXO3arequires functional AMPK. In diabetic patients, FOXO3a is downregulated,which results in glucose level instability.

Glucose related effects in humans have measured an 8 to 10% drop infasting glucose levels in non-diabetic patients, a drop in triglyceridelevels by 10% and a 55% reduction in the amplitude of fasting bloodglucose levels. In diabetic patients, the effect is even more striking,with many patients who were previously on the drug “metformin” beingable to switch with efficacy to 100 mg of enol-oxaloacetic acid and 100mg of ascorbic acid in a once-per-day oral formulation.

The amount of the CR mimetic OAA to use is dependent upon the condition.Metabolic syndrome and diabetes are effectively treated with 100 to1,000 mg enol-oxaloacetic acid in dry form per day, and more preferably100 to 300 mg per day. Such an amount will activate AMPK. The dry powdercan be kept from moisture accumulation by sealing the bottle and using amoisture absorbent such as silicone dioxide. For convenience, the drypowder should be placed in edible capsules or compressed into a pill.Alternately, a salt of oxaloacetic acid can also be used to activateAMPK and treat diabetes; however the salt must either be made up dailyjust prior to use, or part of a biphasic delivery system describedearlier in this application. 100 to 1,000 mg Na-Oxaloacetate iseffective in activating AMPK and treating diabetes. Other mono-valentand tri-valent oxaloacetate salts can also be used to activate AMPK,including, but not limited to, potassium oxaloacetate and chromiumoxaloacetate. Delivery of these salts would be in the same manner anddosage as sodium oxaloacetate.

AMPK activation has been associated with reduced cancer incidence andmetastasis. This is seen with the calorie restriction mimetic and AMPKactivator metformin in meta studies, as well as specific studies withpancreatic and breast cancer cells (Zhuang, Y, et al, Cell cycle arrestin metformin treated breast cancer cells involves activation of AMPK,downregulation of cyclin D1, and requires p27Kipl or p21Cip1, Journal ofMolecular Signaling, (2008)1;3(1):18) (Wang, et. al., Metformin inducesapoptosis of pancreatic cancer cells, World Journal of Gastroenterology,2008, 21:14(47):7192-8.) Calorie Restriction does not prevent cancer,rather, it retards the growth and metastasis of cancer. In a similarmanner, the calorie restriction mimetic OAA inhibits cancer fromreproducing and spreading. uM levels of oxaloacetate in contact withhuman A549 lung cancer tissue in vitro resulted in differential massivedebris within the cancer tissue, but not within normal tissue. Themassive debris resulted in the inability of the cancer tissue toreproduce, even if the cancer tissue was moved away from theoxaloacetate solution for a period of six weeks (Farah, et alDifferential modulation of intracellular energetics in A549 and MRC-5cells, Biomed Sci Instrum 2007 Volume 43, pp 110-5). Farah uses acontact solution of 76 uM, which can be delivered intravenously ororally. If orally, the concentration of OAA must be increased to 1,000to 3,000 mg per day, preferably 500 to 700 mg with three meals duringthe day. Large volumes of OAA prevent the decay of the enzyme hypoxiainducible factor 1 (HIF-1), which leads to increased angiogenesis, whichis undesirable in treating cancer (Lu, H, et. al., ReversibleInacivation of HIF-1 prolyl hydroxylases allows cell metabolism tocontrol basal HIF-1. Journal of Biological Chemistry, 2005280(51):41928-39). In order to prevent HIF-1 from inducing blood vesselformation where it is not needed, ascorbic acid (vitamin C) should beco-administered to the patient with the oxaloacetic acid. The amount ofvitamin C supplemented to the patient can vary from as little as 100 mgupwards to 3,000 mg. Excess Vitamin C is excreted. The combination ofVitamin C and oxaloacetic acid is important because the oxaloacetic acidprevents the reproduction of the cancerous cells, whereas the additionof vitamin C prevents the oxaloacetic acid from stimulating blood vesselformation in existing tumors. While activation of AMPK is required toprevent cell cycling, and the growth and spread of solid tumors, the lowcytotoxicity of OAA does little to kill the existing cells. Thus, inaddition to vitamin C, OAA may be combined with other chemotherapies inorder to destroy existing cancer cells. Optionally, the method caninclude the administration of a chemotherapeutic agent such ascyclophosphamide, chlorambucil, melphalan, estramustine, iphosphamide,prednimustin, busulphan, tiottepa, carmustin, lomustine, methotrexate,azathioprine, mercaptopurine, thioguanine, cytarabine, fluorouracil,vinblastine, vincristine, vindesine, etoposide, teniposide,dactinomucin, doxorubin, dunorubicine, epirubicine, bleomycin,nitomycin, cisplatin, carboplatin, procarbazine, amacrine, mitoxantron,tamoxifen, nilutamid, or aminoglutemide. The compound can beadministered orally, topically, or parenterally. In some aspects of theinvention, the chemotherapeutic agent is administered prior toadministering the oxaloacetate compound. In other aspects, thechemotherapeutic agent is administered after or substantiallycontemporaneously with administering the compound. The cancer can beprimary or metastatic malignant solid tumor disease or a hematologicalmalignancy. If the cancer is a hematological malignancy, it may includeacute and chronic myelogenous leukemia, acute and chronic lymphaticleukemia, multiple myeloma, Waldenstrom's macroglobulinemia, hairy cellleukemia, myelodisplastic syndrome, polycytaemia vera, and essentialthrombocytosis.

AMPK activation has been associated with reduced Alzheimer risk andpotentially with Alzheimer's treatment (Richter, Erik and Ruderman,Neil, “AMPK and the biochemistry of exercise: implications for humanhealth and disease” Biochem. J. (2009) 418, 261-275). Thus OAA may beused for reducing the risk of Alzheimer's disease and for treatment ofthe same. A major advantage of using OAA for neurological diseases isthat OAA can penetrate the blood-brain barrier.

Pharmaceutical Compositions

Pharmaceutical Preparations and Methods of Administration

Oxaloacetate can be administered to an individual at therapeuticallyeffective doses for the prevention or treatment of disorders such asdiabetes, metabolic syndrome, obesity, body weight disorderscardiovascular disease, Alzheimer's disease, and cancer.

As used herein, “oxaloacetate or OAA” includes oxaloacetic acid, thesalt of the acid, or oxaloacetate in a buffered solution as well asmixtures thereof. The term similarly includes oxaloacetate precursorssuch as alpha-ketoglutarate and aspartate.

Effective Dose

A therapeutically effective dose refers to that amount of oxaloacetatesufficient to result in the desired effect such as the amelioration ofsymptoms relating to diabetes, metabolic syndrome, obesity, body weightdisorders cardiovascular disease, Alzheimer's disease, and cancer.

Toxicity and therapeutic efficacy of oxaloacetate can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50 /ED50.The LD50 of alpha-ketoglutarate for mice is above 5 g/kg of body weight.The LD50 of oxaloacetate is above 5 g/kg of body weight. The “noobservable adverse effects level” (NOAEL) in a 90-day sub-chronic ratstudy was 500 mg/kg (the highest dose in the test). Oxaloacetate has avery low toxicity, as would be expected from a chemical involved in theCitric Acid Cycle of every cell.

Toxicity studies of oxaloacetate run in Japan in 1968 on rats indicatesthat levels of oxaloacetate at 83 mg/kg of body weight caused changes inpancreatic islets. Some islets were decreased in size and hyperemic,alpha cells being atrophic, while beta cells were hypertrophic andstained densely. At lower doses, 41 mg/kg of body weight, the pancreasof the rates only demonstrated proliferation and hyperplasia of theislet cells. The liver, hypophysis, adrenals and gonadal glands showedno particular changes (Yoshikawa, Anti-diabetic effect of sodiumoxaloacetate, 1968 Tohoku Journal of Experimental Medicine).

In clinical studies examining the effect of oxaloacetate on diabetes inhumans, 21 diabetic patients received 100 mg to 1,000 mg (2-10 mg/kg ofbody weight). There were no negative side effects. Blood glucose levelsdropped significantly in all patents and urine glucose levels dropped in19 out of the 21 patents (Yoshikawa, Anti-diabetic effect of sodiumoxaloacetate, 1968 Tohoku Journal of Experimental Medicine).

An example of an effective dose of oxaloacetate administered by anintravenous injection is from between about 0.5 mg to about 1 g ofoxaloacetate for each kg of body weight. In a preferred embodiment, theeffective dose of oxaloacetate is between about 2.0 mg and about 40 mgfor each kg of body weight. Due to the acidity of the compound, theeffective dose can be administered in multiple injections over severalhours, or continuously. Effective oral dosing would likewise range fromabout 0.5 mg to about 1 g of oxaloacetate for each kg of body weightwith the preferred effective dosage range between about 2 mg to about 40mg of oxaloacetate for each kg of body weight. For example, an adultmale weighing approximately 80 kg would be administered between about150 mg to about 3.5 g of oxaloacetate orally per day. Dermally, topicalformulations comprising concentrations of about 0.5 to 16 mM ofoxaloacetate are effective. CR studies indicate that restrictingcalories every-other-day yields the same beneficial results as daily CR.Similarly, in some embodiments, oxaloacetate is administeredevery-other-day, as once the genes are activated, the effect lasts forat least a two-day period of time. In other embodiments, oxaloacetate isadministered 3 times per day after each meal.

Formulations

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, oxaloacetateand its physiologically acceptable salts and solvates may be formulatedfor administration by inhalation or insufflation (either through themouth or the nose) or oral, buccal, topical, transdermal, parenteral, orrectal administration. In the case of inhalation, the administration ofoxaloacetate will provide aging benefits directly to lung tissue, evenif the dosage of oxaloacetate administered is less than is needed tobenefit the entire organism. Inhalation of oxaloacetate will delay theon-set of age-related diseases of the lungs and will provide protectionfrom lung diseases.

Oxaloacetate is acidic. The acidity is unlikely to affect organisms thatingest the compound in beneficial amounts as the interior conditions ofthe stomach are also very acidic. The acidity may affect other tissues,including but not limited to the skin or lungs, that may benefit fromthe direct application of oxaloacetate. Therefore, in anotherembodiment, a composition of matter can be created by mixingoxaloacetate with a buffer solution or a base or used as a salt ofoxaloacetate so the delivered compound is not caustic. This will enablehigher concentrations of oxaloacetate to be delivered safely to theorganism, especially if the oxaloacetate is not delivered by oralingestion.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycollate); or wetting agents (e.g., sodium lauryl sulphate).The tablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,non-water solutions, syrups or suspensions, or they may be presented asa dry product for constitution with water or other suitable vehicleimmediately before use (due to decarboxylation concerns). Water acts asa catalyst which allows for the conversion of solid enol-oxaloacetate toconvert to the liquid keto-oxaloacetate form which spontaneouslydecarboxylates into pyruvate and carbon dioxide. Such non-water liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, flavoring, coloring andsweetening agents as appropriate.

While the absorption of oxaloacetate from the digestive tract willincrease the entire organism's oxaloacetate levels, the immediatecontact of oxaloacetate to the cells in the digestive tract willpreferentially be in contact with the digestive tract cells, allowingthe reduction in gastric diseases such as colon cancer, even if theingested amounts of oxaloacetate are insufficient to provide benefit tothe entire organism.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

A topical application is the preferred method of administration ofoxaloacetate for increasing the activity of AMPK directly at the dermisfor the treatment or prevention of skin cancer. The topicalpharmaceutical and cosmetic compositions of the present invention maybemade into a wide variety of product types. These include, but are notlimited to lotions, creams, beach oils, gels, sticks, sprays, ointments,pastes, mousses and cosmetics. These product types may comprise severaltypes of pharmaceutical or cosmetic carrier systems including, but notlimited to solutions, emulsions, gels and solids. The topicalpharmaceutical and cosmetic compositions of the present inventionformulated as solutions typically include a pharmaceutically-acceptableorganic solvent. The terms “pharmaceutically-acceptable organic solvent”refer to a solvent which is capable of having dissolved therein theoxaloacetate, and possesses acceptable safety properties (e.g.,irritation and sensitization characteristics). Examples of a suitablepharmaceutically acceptable organic solvent include, for example,monohydric alcohols, such as ethanol, and polyhydric alcohols, such asglycols. If the topical pharmaceutical and cosmetic compositions of thepresent disclosure are formulated as an aerosol and applied to the skinas a spray-on, a propellant is added to a solution composition.

A type of product that may be formulated from a solution carrier systemis a cream or ointment. An ointment can comprise a simple base of animalor vegetable oils or semi-solid hydrocarbons (oleaginous). An ointmentcan include from about 0.1% to about 2% of a thickening agent. Examplesof suitable thickening agents include: cellulose derivatives (e.g.,methyl cellulose and hydroxy propylmethylcellulose), synthetic highmolecular weight polymers (e.g., carboxyvinyl polymer and polyvinylalcohol), plant hydrocolloids (e.g., karaya gum and tragacanth gum),clay thickeners (e.g., colloidal magnesium aluminum silicate andbentonite), and carboxyvinyl polymers (CARBOPOLS®; sold by B. F.Goodrich Company, such polymers are described in detail in Brown, U.S.Pat. No. 2,798,053, issued Jul. 2, 1975). A more complete disclosure ofthickening agents useful herein can be found in Sagarin, Cosmetics,Science and Technology, 2nd Edition, Vol. 1, pp.72-73 (1972). If thecarrier is formulated as an emulsion, from about 1% to about 10%, forinstance, from about 2% to about 5%, of the carrier system comprises anemulsifier. Suitable emulsifiers include nonionic, anionic or cationicemulsifiers. Exemplary emulsifiers are disclosed in, for example,McCutcheon's Detergents and Emulsifiers, North American Edition, pages317-324 (1986). Preferred emulsifiers are anionic or nonionic, althoughother types can also be employed.

An emulsion carrier system useful in the topical pharmaceutical andcosmetic compositions of the present disclosure is a microemulsioncarrier system. Such a system preferably comprises from about 9% toabout 15% squalane; from about 25% to about 40% silicone oil; from about8% to about 20% of a fatty alcohol; from about 15% to about 30% ofpolyoxyethylene sorbitan mono-fatty acid (commercially available underthe trade name Tweens) or other nonionics; and from about 7% to about20% water. This carrier system is combined with the therapeutic agentsdescribed above, with the oxaloacetate carried in the non-water portion.

The topical pharmaceutical and cosmetic compositions of the presentdisclosure can also include a safe and effective amount of a penetrationenhancing agent. Other conventional skin care product additives may alsobe included in the compositions of the present invention. For example,collagen, elastin, hydrolysates, primrose oil, jojoba oil, epidermalgrowth factor, soybean saponins, mucopolysaccharides, and mixturesthereof may be used. Various vitamins can also be included in thecompositions of the present invention. For example, Vitamin A, andderivatives thereof, Vitamin B2, biotin, pantothenic, Vitamin D, andmixtures thereof can be used.

In yet a further embodiment of the current invention, the oxaloacetatedelivered topically can be mixed with a penetration enhancing agent suchas dimethylsulfoxide (DMSO), combinations of sucrose fatty acid esterswith a sulfoxide or phosphoric oxide, or eugenol, that allows fastermigration of the oxaloacetate into the dermal tissues and then furtherinto deeper cellular tissues, including cellulite tissues wherestimulation of the Sirt1 gene will cause a reduction of fat tissues.

In one embodiment, the disclosed compounds are administered through atopical delivery system. Implantable or injectable polymer matrices, andtransdermal formulations, from which active ingredients are slowlyreleased are also well known and can be used in the disclosed methods.The controlled release components described above can be used as themeans to delivery the disclosed compounds. The compositions can furtherinclude components adapted to improve the stability or effectiveness ofthe applied formulation, such as preservatives, antioxidants, skinpenetration enhancers and sustained release materials. Examples of suchcomponents are described in the following reference works herebyincorporated by reference: Martindale—The Extra Pharmacopoeia(Pharmaceutical Press, London 1993) and Martin (ed.), Remington'sPharmaceutical Sciences.

Controlled release preparations can be achieved by the use of polymersto complex or absorb oxaloacetate. The controlled delivery can beexercised by selecting appropriate macromolecule such as polyesters,polyamino acids, polyvinylpyrrolidone, ethylenevinyl acetate,methylcellulose, carboxymethylcellulose, and protamine sulfate, and theconcentration of these macromolecule as well as the methods ofincorporation are selected in order to control release of activecompound.

In another embodiment, transdermal patches, steady state reservoirssandwiched between an impervious backing and a membrane face, andtransdermal formulations, can also be used to deliver oxaloacetate.Transdermal administration systems are well known in the art. Occlusivetransdermal patches for the administration of an active agent to theskin or mucosa are described in U.S. Pat. Nos. 4,573,996, 4,597,961 and4,839,174, which are hereby incorporated by reference. One type oftransdermal patch is a polymer matrix in which the active agent isdissolved in a polymer matrix through which the active ingredientdiffuses to the skin. Such transdermal patches are disclosed in U.S.Pat. Nos. 4,839,174, 4,908,213 and 4,943,435, which are herebyincorporated by reference. In one embodiment, the steady state reservoircarries doses of oxaloacetate in doses from about 2 mg to 40 mg per day.

Present transdermal patch systems are designed to deliver smaller dosesover longer periods of time, up to days and weeks. A rate-controllingouter microporous membrane, or micropockets of the disclosedoxaloacetate dispersed throughout a silicone polymer matrix, can be usedto control the release rate. Such rate-controlling means are describedin U.S. Pat. No. 5,676,969, which is hereby incorporated by reference.In another embodiment, the oxaloacetate is released from the patch intothe skin of the patient in about 20-30 minutes or less.

These transdermal patches and formulations can be used with or withoutuse of a penetration enhancer such as dimethylsulfoxide (DMSO),combinations of sucrose fatty acid esters with a sulfoxide or phosphoricoxide, or eugenol. The use of electrolytic transdermal patches is alsowithin the scope of the methods disclosed herein. Electrolytictransdermal patches are described in U.S. Pat. Nos. 5,474,527,5,336,168, and 5,328,454, the entire contents of which are herebyincorporated by reference.

Oxaloacetate may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. The injectedoxaloacetate can be mixed with other beneficial agents prior toinjection including but not limited to antibiotics and othermedications, saline solutions, blood plasma, and other fluids. Immediatecontact of elevated levels of oxaloacetate with the vascular systemcells will result in the reduction in age-related diseases such ashardening of the arteries, even if the amounts of oxaloacetate areinsufficient to provide age-related benefits to the entire organism.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before immediate use.

Oxaloacetate may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, oxaloacetate mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

In yet still another embodiment, oxaloacetate can be mixed with animalfoods for the prevention or treatment of disorders such as diabetes,metabolic syndrome, obesity, cardiovascular disease, Alzheimer'sdisease, and cancer in animals. Oxaloacetate can either be formulated aspart of the animal food or administered separately as a supplement tothe animal's food. As those skilled in the art know, dry pet foods,typically dry dog foods, normally contain protein, fat, fiber, non-fibercarbohydrates, minerals, vitamins and moisture components. For example,as major ingredients, there are typically one or two cereal grains,generally corn, wheat and/or rice. In addition, for a protein sourcethey may contain poultry meal, by-product meat, meat and bone meal, orother animal or fish meal by-products. At times as well, grain proteinsupplements such as corn gluten, soybean meal or other oil seed mealscan be added. In addition to an effective amount of oxaloacetate ofbetween about 0.01% to 0.1% by weight of the chow, animal chow of thepresent invention additionally includes the following: typical nutrientcontent in the food dry matter includes crude protein from 14% to 50%,usually 20% to 25%; crude fat from 5% to 25%; and crude fiber usually ispresent in the range of from about 3% to 14%, usually about 5% to 7%,with the total mineral or ash content being within the range of 3% to10%, usually 4% to 7%. The important point is not the preciseformulation of the pet food, since many conventional and satisfactoryones for use in conjunction with the present invention are available onthe market. Rather, the key to success is that a sufficient amount ofoxaloacetate component be added to pet food rations, whicheverformulation is used, to provide the oxaloacetate activity level at theranges necessary for AMPK activation in order to support the preventionor treatment of disorders such as diabetes, metabolic syndrome, obesity,cardiovascular disease, Alzheimer's disease, and cancer in animals.

Uses and Indications

Based on the inventors' findings (see examples below) and other data inthe literature that relate to the benefits of AMPK activation referencedherein, it is contemplated that the compounds and pharmaceuticalcompositions presented herein are used as prophylactic and/ortherapeutic agents for various conditions, and particularly in theprevention and/or treatment of metabolic syndrome, pre-diabetes, insulinresistance, type 2 diabetes, and/or dyslipidemia. It should beappreciated that the term “treated” or “treatment” where used inconjunction with a medical condition refers to at least one of aresolution and/or improvement in clinical parameters of clinicallyabnormal values, and/or to an improvement in subjective feeling of apatient diagnosed with the condition.

Viewed from another perspective, it is also contemplated that variousbenefits may be derived from administration of the compounds andpharmaceutical compositions presented herein, and especiallycontemplated benefits relate to prevention, amelioration, and/ortreatment of diseases or conditions associated with activation of AMPK,and the following provides exemplary guidance on contemplated benefits.

Hyperglycemia

It has recently been reported that therapeutic doses of metforminincrease AMPK activity in vivo in subjects with type 2 diabetes(Diabetes, 51(7): 2074-81, 2002). Metformin treatment for 10 weekssignificantly increased AMPK alpha 2 activity in the skeletal muscle,and this was associated with increased phosphorylation of AMPK on Thr172and decreased acetyl-CoA carboxylase-2 activity. The increase in AMPKalpha 2 activity was likely due to a change in muscle energy statusbecause ATP and phosphocreatine concentrations were lower aftermetformin treatment. Metformin-induced increases in AMPK activity wereassociated with higher rates of glucose disposal and muscle glycogenconcentrations. These findings suggest that the metabolic effects ofmetformin in subjects with type 2 diabetes may be mediated by theactivation of AMPK alpha 2. Given the hypoglycemic effect imparted bythe activation of AMPK, administration of contemplated pharmaceuticalproducts to increase AMPK activity may be useful to lower blood glucoseconcentrations by decreasing hepatic glucose production and increasingglucose disposal in skeletal muscle.

Athletic Performance

AMPK activation leads to PGC-lalpha activation which leads tomitochondrial biosynthesis (Lopez-Lluch, et al. Mitochondrial Biogenesisand Healthy Aging, Expermental Gerontology, 2009 September;43(9):813-819.doi:10.1016/j.exger.2008.06.014.) Increasing mitochondrialbiosynthesis will lead to increased mitochondrial density in the musclecells. Increased mitochondrial density will increase athleticperformance in terms of muscle strength and endurance.

Reduced Insulin Sensitivity

Conditions and disorders associated with diminished insulin sensitivityof muscle glucose transport may be treated by administration ofcontemplated compounds. Various reports suggest that increase in insulinsensitivity of muscle glucose transport following exercise is mediatedby activation of AMPK. Thus, ingestion of contemplated pharmaceuticalproducts is thought to provide increased insulin sensitivity of muscleglucose transport.

Insulin Resistance Syndrome

Insulin resistance syndrome is associated with obesity, type 2 diabetes,and muscle paralysis (see e.g., WO 01/97816 A1 and WO 01/93874 A1).Insulin resistance syndrome is also associated with several risk factorsfor cardiovascular disease. In view of numerous papers suggesting thatactivating AMPK improves glucose tolerance, improves the lipid profile,and reduces systolic blood pressure, ingestion of contemplatedpharmaceutical products to increase AMPK activity is deemed useful toreduce metabolic disturbances and/or to lower blood pressurecharacteristic of insulin resistance syndrome.

Insufficient Glucose Uptake in Muscle Cells

It has been observed that exercise and/or electrical stimulation ofvarious muscles increases AMPK activity, and consequently increasesglucose uptake. Based on these observations, it has been hypothesizedthat muscle contraction plays a role in stimulating glucose uptake inmuscle, where one mechanism underlying increased uptake stems fromactivated AMPK increasing GLUT-4 translocation from microvesicles tosarcolemmal membranes in muscle.

Based on the inventors' observation that compounds with cytokininactivity increase AMPK activity, it should be recognized thatcontemplated pharmaceutical products may be beneficial in enhancingglucose uptake into muscle cells (as well as being beneficial inameliorating disorders that are characterized by decreased glucoseuptake in muscle cells, or that are exacerbated by the effects ofdecreased glucose uptake in muscle cells).

Insulin Oversecretion

It is generally accepted in the art that activated AMPK inhibits insulinsecretion, and as contemplated compounds were demonstrated to activateAMPK, it should be recognized that treatment with such compounds shouldprovide a significant reduction in insulin secretion. Consequently,conditions associated with oversecretion of insulin may benefit fromingestion of contemplated pharmaceutical products.

Dyslipidemia

Hepatic acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoAreductase (HMGR) are two targets for the AMPK system, catalyzing the keyregulatory steps in fatty acid and sterol synthesis, respectively(Winder et al, Am J Physiol, 2777: E1-10, 1999, the entirety of which isherein incorporated by reference.) Activation of AMPK serves to inhibitboth these lipid biosynthetic pathways, as well as triglyceridesynthesis. Moreover, it is contemplated that activated AMPK inhibits theL-type pyruvate kinase and fatty acid synthase gene expression.

Reduction of activity of ACC in the liver cell also leads to decreasesin the concentration of the product of ACC, i.e., malonyl-CoA, which hasmarked effects on fatty acid oxidation. Malonyl-CoA is a potentinhibitor of carnitine palmitoyltransferase-1 (CPT-1), the “gatekeeper”for entry of fatty acids into the mitochondria. In the liver, fatty acidoxidation can be considered to be an essential component of the pathwayfor synthesis of ketone bodies: increases in fatty acid oxidation leadto increased hepatic ketogenesis. It is therefore contemplated thatadministration of contemplated compounds at a concentration effective toactivate AMPK in the liver would result in decreases in fatty acid,triglyceride, and sterol synthesis and increases in fatty acid oxidationand ketogenesis. Viewed from another perspective, contemplatedpharmaceutical products may be useful to increase AMPK activity andthereby reduce fatty acid synthesis, sterol synthesis, triglyceridesynthesis and fatty acid synthase gene expression. Of additional benefitis also the AMPK-mediated increase in activity in fatty acid oxidationand ketogenesis, where increased ketogenesis is desired.

Obesity

Hormone-sensitive lipase (HSL) is a target for AMPK in adipose tissue.Activation of AMPK has been shown to inhibit lipogenesis byphosphorylation of ACC and also to inhibit isoprenaline-stimulatedlipolysis. Thus, contemplated pharmaceutical products may help reduce oreven abolish lipogenesis and/or increase isoprenaline-stimulatedlipolysis. Thus, and given the inhibitory role for AMPK in the processof adipose differentiation, it should be recognized that contemplatedpharmaceutical products will likely inhibit adipogenesis.

Reduction in Fat accumulation after dieting

Activation of AMPK has been shown to inhibit lipogenesis, which isincreased after ending a diet. The increase in lipogenesis leads torapid increases in weight, often referred to as the “diet reboundeffect”. This rebound effect can be reduced or eliminated with OAAsupplementation.

Modulation of Stability of Selected mRNA Species

HuR is an RNA binding protein that functions to stabilize a variety oftarget mRNA transcripts, including those encoding p21, cyclinA andcyclinB1. It has been shown that the presence of activated AMPK resultsin reduced levels of cytoplasmic HuR, and in turn, in reducedconcentrations and half-lives of mRNA encoding p21, cyclinA and cyclinB1(see e.g., Mol Cell Biol, 22(10):345-36, 20002, which is incorporatedherein by reference). Thus, treatment with contemplated compounds willincrease AMPK activity, and thus reduce levels of cytoplasmic HuR, whichis thought to reduce concentrations/half-lives of a variety of targetmRNA transcripts, including those ending p21, cyclinA and cyclinB1.

Premature Apoptosis

Activated AMPK has been shown to provide protection againstglucocorticoid-induced apoptosis and to restore cell viability andinhibit DNA laddering in dexamethasone-treated thymocytes (see e.g.,Biochem Biophys Res Commun, 243(3):821-6, 1998, which is incorporatedherein by reference). Furthermore, activated AMPK has been shown toprovide protection against dexamethasone-induced activation of caspase3-like enzymes, which are believed to play a pivotal role in apoptoticcell death. Thus, treatment with contemplated compounds to increase AMPKactivity may provide protection against glucocorticoid-inducedapoptosis.

Ischemia

Conditions and disorders associated with AMPK regulation of cellularresponses to stresses, including ischemia, are among those treatable byadministering a composition comprising a compound that activates AMPK.In several non-vascular tissues, AMPK appears to modulate the cellularresponse to stresses such as ischemia. In liver and muscle, AMPKphosphorylates and inhibits acetyl CoA carboxylase (ACC), leading to anincrease in fatty acid oxidation; in muscle, AMPK activation isassociated with an increase in glucose transport. Furthermore,incubation of human umbilical vein endothelial cells (HUVEC) with anAMPK activator has been shown to cause a 5-fold activation of AMPK,which was accompanied by a 70% decrease in ACC activity and a 2-foldincrease in fatty acid oxidation. (Biochem Biophys Res Commun,265(1):112-5, 1999, which is incorporated herein by reference). However,in this same study, glucose uptake and glycolysis, the dominantenergy-producing pathway in HUVEC, were diminished by 40-60% under theseconditions. Despite this, cellular ATP levels were increased by 35%.Thus, treatment with contemplated compounds to increase AMPK activity isexpected to result in major alterations in endothelial cell energybalance, which are useful in providing protection against cellularstresses in conditions including ischemia.

Metabolic and Excitotoxic Insults

It is well known in the art that the brain has a high metabolic rate andis relatively sensitive to changes in the supply of glucose and oxygen.The expression of AMPK in embryonic and adult brain and its role inmodifying neuronal survival under conditions of cellular stress havebeen investigated (J Mol Neurosci, 17(1): 45-58, 2001). Catalytic (alpha1 and alpha 2) and noncatalytic (beta 2 and gamma 1) subunits of AMPKare present at high levels in embryonic hippocampal neurons in vivo andin cell culture. In the adult brain, the catalytic subunits alpha 1 andalpha 2 are present in neurons throughout the brain. The AMPK-activatingagent AICAR protected hippocampal neurons against death induced byglucose deprivation, chemical hypoxia, and exposure to glutamate andamyloid beta-peptide. Suppression of levels of the AMPK alpha 1 andalpha 2 subunits using antisense technology resulted in enhancedneuronal death following glucose deprivation, and abolished theneuroprotective effect of AICAR. Thus, given the role of AMPK activationin modifying neuronal survival under conditions of cellular stress,treatment with contemplated compounds to increase AMPK activity isthought to provide protection of neurons against metabolic andexcitotoxic insults.

Similarly, conditions and disorders associated with hypoxia may betreated using contemplated compounds. AMPK is believed to play a role inregulating ketone body production by astrocytes. (J Neurochem, 73(4):1674-82, 1999). Incubation of astrocytes with AICAR has been shown tostimulate both ketogenesis from palmitate and carnitinepalmitoyltransferase I concomitant to a decrease of intracellularmalonyl-CoA levels and an inhibition of acetyl-CoA carboxylase/fattyacid synthesis and 3-hydroxy-3-methylglutaryl-CoA reductase/cholesterolsynthesis. Moreover, microdialysis experiments have shown AICAR tostimulate brain ketogenesis markedly. Incubation of astrocytes withazide has been shown to lead to a remarkable drop of fatty acidbeta-oxidation. However, activation of AMPK during hypoxia was shown tocompensate the depression of beta-oxidation, thereby sustaining ketonebody production. The effect is believed to rely on the followingcascade: hypoxia leads to an increase of the AMP/ATP ratio, whichtriggers AMPK stimulation, which in turn results in acetyl-CoAcarboxylase inhibition. Consequently, malonyl-CoA concentrationdecreasesm and carnitine palmitoyltransferase I is activated, thusenhancing ketogenesis. Furthermore, incubation of neurons with azide hasbeen shown to blunt lactate oxidation, but not 3-hydroxybutyrateoxidation. Thus, given the role of AMPK activation in regulating ketonebody production by astrocytes, treatment with contemplated compounds toincrease AMPK activity is useful in promoting astrocytes to produceketone bodies as a substrate for neuronal oxidative metabolism duringhypoxia.

Hepatic Ischemia-Reperfusion

Hepatic ischemia-reperfusion (I/R) injury associated with livertransplantation and hepatic resections may be reduced by administering acomposition comprising a compound that activates AMPK. Preconditioningis known to preserve energy metabolism in liver during sustainedischemia. A study has been reported that investigates: 1) whetherpreconditioning induces AMPK activation; and 2) if AMPK activation leadsto ATP preservation and reduced lactate accumulation during prolongedischemia and its relationship with NO (Hepatology, 34(6): 1164-73,2001). Preconditioning was reported to activate AMPK and concomitantlyreduce ATP degradation, lactate accumulation, and hepatic injury. Theadministration of an AMPK activator, AICAR, before ischemia simulatedthe benefits of preconditioning on energy metabolism and hepatic injury.The inhibition of AMPK abolished the protective effects ofpreconditioning. The effect of AMPK on energy metabolism was independentof NO because the inhibition of NO synthesis in the preconditioned groupand the administration of the NO donor before ischemia, or to thepreconditioned group with previous inhibition of AMPK, had no effect onenergy metabolism. Thus, given the role of AMPK activation in theprotective effect against ischemia, treatment with contemplatedcompounds to increase AMPK activity is contemplated for surgical andpharmacological strategies aimed at reducing hepatic I/R injury.

It is well established that nutrient deprivation activates AMPK (supra),and that tumors in a relatively early stage are dependent on nutrientdiffusion. Thus, when a tumor reaches a critical mass, AMPK will beactivated in at least some cells due to lack of glucose and other growthfactors. Consequently, the inventors contemplate that contemplatedanticytokinins may be employed to block energy salvage pathways of tumorcells (see e.g., Oncogene. 2002 Sep. 5; 21(39):6082-90: Critical rolesof AMP-activated protein kinase in constitutive tolerance of cancercells to nutrient deprivation and tumor formation by Kato et al.).

Therefore, it should be appreciated that contemplated pharmaceuticalcompositions and contemplated compounds may especially beneficial to aperson to (1) reduce fatty acid synthesis, sterol synthesis,triglyceride synthesis and fatty acid synthase gene expression, (2)ameliorate one or more conditions or disorders that are characterized byelevations in one or more of the pathways or mechanisms involved infatty acid synthesis, sterol synthesis, triglyceride synthesis and fattyacid synthase gene expression, (3) increase fatty acid oxidation andketogenesis, (4) inhibit lipogenesis and/or isoprenaline-stimulatedlipolysis, (5) ameliorate one or more conditions or disorders that arecharacterized by elevations in one or both of lipogenesis andisoprenaline-stimulated lipolysis pathways, or that are exacerbated bythe elevations in one or both of these pathways, (6) decrease insulinsecretion, (7) ameliorate one or more a conditions or disorders that arecharacterized by elevated insulin secretion, or that are exacerbated byinsulin secretion, (8) enhance glucose uptake in muscle cells, (9)ameliorate one or more conditions or disorders that are characterized bydecreased glucose uptake in muscle cells, or that are exacerbated by theeffects of decreased glucose uptake in muscle cells, (10) inhibitadipogenesis, (11) ameliorate one or more conditions or disorders thatare characterized by increased adipogenesis, or that are exacerbated byadipogenesis, (12) increase insulin sensitivity of muscle glucosetransport, (13) lower blood glucose concentrations by decreasing hepaticglucose production and/or increasing glucose disposal in skeletalmuscle, and/or (14) ameliorate one or more conditions or disordersassociated with insulin resistance syndrome through improving glucosetolerance, improving lipid profile or reducing systolic blood pressure.

It should be especially appreciated that traditional long term metformintherapy often requires concurrent supplementation with calcium carbonateto prevent the adverse impact of metformin administration on vitamin B12 absorption. It has been postulated that the hydrophobic tail ofbiguanides, such as metformin, extends into the hydrophobic core ofmembranes, thereby adding a positive charge to the surface of themembrane, which acts to displace divalent cations (see e.g., Bauman etal. (Diabetes Care 2000; 23:1227-31)), which in turn negatively affectsbinding of the B 12-intrinsic factor complex to the ileal cell surfacereceptors. Such adverse consequences are not expected using OAA as theyare significantly structurally different from biguanides.

Consequently, the inventor contemplates a method of modulating glucosemetabolism in a mammal in which in one step a contemplatedcompound/pharmaceutical composition is administered to a the mammal at adosage effective to modulate glucose metabolism in the mammal. Inespecially contemplated aspects, the mammal is a human and diagnosedwith metabolic syndrome, pre-diabetes, insulin resistance, type-2diabetes, and/or dyslipidemia. Additionally, or alternatively,contemplated compounds/pharmaceutical compositions may also beprophylactically administered to prevent or delay onset or progressionof metabolic syndrome, pre-diabetes, insulin resistance, type-2diabetes, and/or dyslipidemia. While not limiting to the inventivesubject matter, the inventors contemplate that such treatment may be dueto an increase in glucose uptake into a muscle cell (or other cell),and/or due to a decrease in gluconeogenesis in a hepatocyte. Withrespect to the hepatocyte, and while not limiting to the inventivesubject matter, the inventors contemplate that the compounds presentedherein will directly or indirectly affect activity of the glucocorticoidreceptor, PEPCK (phosphoenolpyruvate carboxykinase), the glucagonreceptor, and/or glucose-6-phosphatase. From a genomic standpoint,activation of AMPK also increases the expression of the FOXO3a gene,associated with increasing glucose homeostasis.

Similarly, in further preferred aspects, the inventors contemplate amethod of modulating lipid metabolism in a mammal in which in one step acontemplated compound/pharmaceutical composition is administered to athe mammal at a dosage effective to modulate glucose metabolism in themammal. Such methods may advantageously be employed to treat or preventmetabolic syndrome and/or dyslipidemia, and may also be employed todecrease at least one of total serum cholesterol, serum LDL-cholesterol,and serum triglycerides.

Viewed from another perspective, the inventors also contemplate a methodof treating a condition in a mammal, wherein the condition is associatedwith a dysregulation of at least one of AMPK. In such methods, acontemplated compound/pharmaceutical composition is administered to themammal at a dosage effective to activate AMPK. Among other conditions,especially contemplated conditions for such methods includecardiovascular disease, type 2 diabetes, Alzheimer's disease and aneoplastic disease.

EXAMPLES Example 1

A non-diabetic subject is given 100 mg anhydrous enol-oxaloacetic acidin solid form for a period of 6 months to reduce blood triglycerides andglucose levels. AMPK is activated and the treatment is effective.

Feb-07 Aug-07 % Change Glucose Fasting (mg/dl) 97 89 8.25% Decrease(Improvement) Lipid Panel Triglyceride (mg/dl) 112 102 8.93% Decrease(Improvement) Total Cholesterol (mg/dl) 202 203 HDL Ser Cholesterol(mg/dl) 42 42 LDL Cholesterol (mg/dl) 138 138 Glomeruler Filtration Rate(GFR) Bun-Ser (mg/dl) 14 12 Albumin (g/dl) 4.4 4.2 Creatinine (mg/dl) 10.9 Electrolyte Panel Na (Sodium) (mEq/L) 144 143 Cl (Chloride) (mEq/L)110 108 K (Potassium) (mEq/L) 4.6 4.5 CO2 (Carbon Dioxide) 27 29 (mEq/L)ALK P′TASE (IU/L) 36 37 AST (SGOT) (IU/L) 16 15 TSH (uIU/ml) 1.41 1.32Lipase (u/l) 17 19

Example 2

A patient is given 100 mg anhydrous enol-oxaloacetic acid daily in solidform and blood glucose levels are tracked for several weeks. Theamplitude of the patient's swings in fasting glucose decrease by 55% dueto improved glucose homeostasis. AMPK is activated and overall fastingglucose levels drop by 8%. The data is shown in Graph 3.

Example 3

A patient is given 100 mg anhydrous enol-oxaloacetate daily in solidform and continues with his normal routine. AMPK is activated and thepatient drops in weight from 258 to 234 pounds in a three month period.The patient is no longer obese.

Starting Weight Feb 2007 Ending Weight May 2007 Percent Reduction 258234 9.3%

Example 4

A patient successfully loses 24 pounds. In order to maintain the weightloss, the patient is given 100 mg anhydrous enol-oxaloacetic acid dailyin solid form. AMPK is activated which down regulates human homologuesof the following genes in the liver; Acaal, Cnbp, Fasn, Idil, Ndufabl,Pcx, Sc5d, all of which reduces the body's ability to create and storefat. The patient does not regain the weight lost during a one-yearperiod after the diet.

Example 5

A patient is diagnosed with hypertension. The patient is given 100 mganhydrous enol-oxaloacetic acid daily in solid form for three months.The patient's blood pressure decreases by 10%.

Example 6

A patient has a family history of colon cancer. The patient is given 200mg anhydrous enol-oxaloacetic acid and 200 mg Vitamin C in solid form inorder to reduce the development of cancerous polyps into colon cancer.After a three year interval between colonoscopies, the incidence ofpre-cancerous polyps is reduced by 50%, despite the increase in age ofthe patient and the typical correlation between increasing age andincreasing polyp incidence.

Example 7

A patient is diagnosed with lung cancer. The patient is given 500 mganhydrous enol-oxaloacetic acid with each meal along with 500 mg VitaminC in solid form in addition to chemotherapy. The patient has asuccessful recovery from cancer. The patient is placed on a maintenancedose of 200 mg anhydrous enol-oxaloacetic acid and 200 mg Vitamin C perday to reduce risk of reoccurrence.

Example 7

A patient has a family history of Alzheimer's disease. The patient isadministered 200 mg anhydrous enol-oxaloacetic acid and 200 mg Vitamin Cper day to activate AMPK and delay incidence of Alzheimer's disease.

Example 8

Here I report a case study on a 73 year old Hungarian woman, weight of89 kg, with a history of difficult to treat Type 2 diabetes. The trialwas a “grass-roots” look at glucose levels performed by the patient witha glucose test strip meter under normal living conditions. Theunsolicited data was supplied to us as the developer of the nutritionalsupplement product. Unfortunately, no other measurements were taken bythe patient other than blood glucose levels, but the number of readingstaken by the patient is impressive, and does show statisticallysignificant results. At the start of the study, the woman was on thefollowing medications:

-   -   Diaprel MR/(80 mg)-2 per day—Extended release Gliclazide (80        mg), a once per day diabetic drug    -   Pentoxyl-EP (400 mg)—1 per day—Contains pentoxifylline, used for        intermittent Claudication (A symptom complex characterized by        leg pain and weakness brought on by walking, with the        disappearance of the symptoms following a brief rest).    -   Merckformin (1,000 mg)—1 per day—(Metformin, Glucophage)—used        for Type 2 diabetes    -   Glycerine sol (0.5 d1) After dinner    -   Avandamet—1 per day—a combination of metformin and rosiglitazone        used for diabetes

Her fasting glucose levels fluctuated from the 8 to 11 mmol/L andglucose levels after a meal increased up to 12 mmol/L. In addition toher current medicines, the patient self started 100 mg/ day stabilizedoxaloacetic acid with 100 mg/day Vitamin C (combined in 1 capsule).During the study the patient increased the use of combination to twocapsules, then three capsules per day. By the end of the 70 day studythe patient's fasting glucose levels had dropped to a range between 7and 8 mmol/L, and glucose levels after a meal remained more consistentin the 7 to 8.5 mmol/L range, levels not achievable with the threeprescription medications the patient was consuming. Using linear trendanalysis, the patient's fasting glucose levels dropped 23% from thestart of the test to the end of the test. Her glucose levels after ameal dropped 34.5%, indicating a major improvement in glucose managementand glucose tolerance. Comparison of the various glucose levels in thefirst half of the trial versus the second half of the trail yields a pvalue of <0.001, indicating a very significant difference between thefirst half and second half of the trial.

The reduction in glucose levels occurred despite a reduction in theamount of Mercformin (Metformin, Glucophage) from 1,000 mg/day to 850mg/day during the study. The patient also stopped using Pentoxyl-EP andglycerol by the end of the study. The data from this case study is shownas FIG. 1. Improvement in this patient's glucose levels and glucosetolerance with stabilized oxaloacetic acid, an over-the-counter dietarysupplement, was shown to successfully support proper glucosefunctioning.

Example 9

AMPK activation is necessary to increase lifespan of C. elegans whenexposed to 8 mM concentrations of oxaloacetate. The increase in lifespanaverages approximately 25%, p<0.001. In C. elegans that contain adysfunctional AMPK gene (dysfunctional in the aak-2 subunit), AMPK isnot activated by oxaloacetate, and there is no increase in lifespan.

The foregoing description details certain embodiments of the invention.It will be appreciated, however, that no matter how detailed theforegoing appears in text, the invention can be practiced in many ways.As is also stated above, it should be noted that the use of particularterminology when describing certain features or aspects of the inventionshould not be taken to imply that the terminology is being redefinedherein to be restricted to including any specific characteristics of thefeatures or aspects of the invention with which that terminology isassociated. The scope of the invention should therefore be construed inaccordance with the appended claims and any equivalents thereof.Additionally, throughout this application, various publications havebeen referenced. The disclosures in these publications are incorporatedherein by reference in order to more fully describe the state of theart.

The invention claimed is:
 1. A composition comprising a therapeuticallyeffective amount from about 100 mg to about 3,000 mg anhydrousenol-oxaloacetic acid.
 2. The composition of claim 1, wherein thecomposition comprises less than about 2% water.
 3. The composition ofclaim 2, wherein the composition further comprises from about 100 mg toabout 1,000 mg vitamin C.
 4. The composition of claim 1, wherein thecomposition comprises less than about 1% water.
 5. The composition ofclaim 4, wherein the composition further comprises from about 100 mg toabout 1,000 mg vitamin C.
 6. The composition of claim 1, wherein thecomposition further comprises from about 100 mg to about 1,000 mgvitamin C.
 7. The composition of claim 1, wherein the compositioncomprises from about 100 mg to about 300 mg anhydrous enol-oxaloaceticacid.
 8. The composition of claim 7, wherein the composition comprisesless than about 2% water.
 9. The composition claim 8, wherein thecomposition further comprises from about 100 mg to about 1000 mg vitaminC.
 10. The composition of claim 7, wherein the composition comprisesless than about 1% water.
 11. The composition of claim 10, wherein thecomposition further comprises from about 100 mg to about 1000 mg vitaminC.
 12. The composition of claim 7, wherein the composition furthercomprises from about 100 mg to about 1000 mg vitamin C.
 13. Thecomposition of claim 7, wherein the composition comprises about 100 mganhydrous enol-oxaloacetic acid.
 14. The composition of claim 13,wherein the composition comprises less than about 2% water.
 15. Thecomposition of claim 14, wherein the composition further comprises fromabout 100 mg to about 1000 mg vitamin C.
 16. The composition of claim13, wherein the composition comprises less than about 1% water.
 17. Thecomposition of claim 16, wherein the composition further comprises fromabout 100 mg to about 1000 mg vitamin C.
 18. The composition of any oneof claim 13, wherein the composition further comprises from about 100 mgto about 1000 mg vitamin C.
 19. The composition of claim 18, wherein thecomposition comprises about 100 mg vitamin C.